Ceramides (CER) are involved in alcohol induced neuroinflammation. In a mouse model of chronic alcohol exposure, 16 CER and 18 sphingomyelin (SM) concentrations from whole brain lipid extracts were measured using ESI mass spectrometry. All 18 CER concentrations in alcohol exposed adults increased significantly (range: 25607%); in juveniles, 6 CER decreased (range: 9 to -37%). In contrast, only three SM decreased in adult and one increased significantly in juvenile. Next, regional identification at 50 m spatial resolution from coronal sections was obtained with matrix implanted laser desorption/ionization mass spectrometry imaging (MILDI-MSI) by implanting silver nanoparticulate matrices followed by focused laser desorption. Most of the CER and SM quantified in whole brain extracts were detected in MILDI images. Coronal sections from three brain levels show qualitative regional changes in CER-SM ion intensities, as a function of group and brain region, in cortex, striatum, accumbens, habenula, and hippocampus. Highly correlated changes in certain white matter CER-SM pairs occur in regions across all groups, including the hippocampus and the lateral (but not medial) cerebellar cortex of adult mice.